长江鲟科鱼类尿液成分中L-色氨酸的检测分析
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摘要
采用高效液相色谱-荧光法(HPLC-FLD)建立了中华鲟和长江鲟尿液中L-色氨酸的测定方法。中华鲟和长江鲟尿液样品由V(甲醇)∶V(乙腈)=1∶1提取,离心后,上清液经ENVI-18固相萃取小柱净化,利用symmetry@C18色谱柱(250 mm×4.6 mm,5μm)进行分离,以乙腈-1%(体积分数)甲酸水溶液(10∶90,体积分数)为流动相等度洗脱,在流速1.0 mL/min,荧光波长λ_(ex)=285 nm,λ_(em)=353 nm条件下进行检测。结果显示:L-色氨酸标准品的峰面积与其质量浓度在0.010～2.000μg/mL范围内线性关系良好,线性回归方程为y=247.27x-0.125 5(R~2=1)。L-色氨酸的检出限(LOD)为0.005μg/mL,定量限(LOQ)为0.020μg/mL。L-色氨酸空白加标回收率为82.2%～102.1%,样品基质加标回收率为85.6%～95.1%,相对标准偏差(RSD)均小于5.7%。本方法有良好的回收率和精密度,且步骤简便,经济省时,可用于中华鲟和长江鲟尿液L-色氨酸的检测,为研究L-色氨酸在濒危鲟鱼中华鲟和长江鲟信息交流方面的作用奠定方法学基础。
A method of high performance liquid chromatography-fluorescence spectroscopy(HPLC-FLD) was used to determinate L-tryptophan in urine of Acipenser sinensis and A. dabryanus. The samples were extracted by methanol and acetonitrile, by centrifugation to get supernatant, and purified by an ENVI-18 solid-phase extraction column. The acetonitrile-1%(volume fraction) formic acid aqueous solution(10∶90, volume fraction) was used as the mobile phases, the extracts were separated on a symmetry@ C18 chromatographic column(250 mm ×4.6 mm, 5 μm). The samples were determinated under the condition of the flow rate of 1.0 mL/min, with the fluorescence wavelength of λ_(ex)=285 nm, λ_(em)=353 nm. The results indicated that the significant linear relationships between peak areas and mass concentrations of the analytes were obtained in the range of 0.010-2.000 μg/mL(R~2=1), and linear regression equation was y = 247.27x-0.125 5. The limits of detection(LOD) and limits of quantification(LOQ) of L-tryptophan were 0.005 μg/mL and 0.020 μg/mL. The recoveries of blank matrix and sample substrate with standard samples were 82.2%-102.1% and 85.6%-95.1%, respectively, with less than 5.7% of the relative standard deviations(RSD). This method has some obvious advantage such as easy operation, time saving, high recoveries and high accuracies. It will be valuable to analyze L-tryptophan in urine of A. sinensis and A. dabryanus. A methodology basis is provided to study the role of L-tryptophan in information exchange of A. sinensis and A. dabryanus.
A method of high performance liquid chromatography-fluorescence spectroscopy(HPLC-FLD) was used to determinate L-tryptophan in urine of Acipenser sinensis and A. dabryanus. The samples were extracted by methanol and acetonitrile, by centrifugation to get supernatant, and purified by an ENVI-18 solid-phase extraction column. The acetonitrile-1%(volume fraction) formic acid aqueous solution(10∶90, volume fraction) was used as the mobile phases, the extracts were separated on a symmetry@ C18 chromatographic column(250 mm ×4.6 mm, 5 μm). The samples were determinated under the condition of the flow rate of 1.0 mL/min, with the fluorescence wavelength of λ_(ex)=285 nm, λ_(em)=353 nm. The results indicated that the significant linear relationships between peak areas and mass concentrations of the analytes were obtained in the range of 0.010-2.000 μg/mL(R~2=1), and linear regression equation was y = 247.27x-0.125 5. The limits of detection(LOD) and limits of quantification(LOQ) of L-tryptophan were 0.005 μg/mL and 0.020 μg/mL. The recoveries of blank matrix and sample substrate with standard samples were 82.2%-102.1% and 85.6%-95.1%, respectively, with less than 5.7% of the relative standard deviations(RSD). This method has some obvious advantage such as easy operation, time saving, high recoveries and high accuracies. It will be valuable to analyze L-tryptophan in urine of A. sinensis and A. dabryanus. A methodology basis is provided to study the role of L-tryptophan in information exchange of A. sinensis and A. dabryanus.
引文
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[3] 赵明江,周小秋.色氨酸对应激诱导鱼类皮质醇分泌的影响[J].水产科学,2007,26(7):420-422.ZHAO M J,ZHOU X Q.The effect of dietary tryptophan on the stress-induced secretion of cortisol[J].Fisheries Science,2007,26(7):420-422.
[4] YAMBE H,KITAMURA S,KAMIO M,et al.L-Kynurenine,an amino acid identified as a sex pheromone in the urine of ovulated female masu salmon[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(42):15370-15374.
[5] 赖晓健,洪万树,张其永.鱼类嗅觉系统和性信息素受体的研究进展[J].动物学杂志,2103,48(2):298-305.LAI X J,HONG W S,ZHANG Q Y.Research progresses in fish olfactory system and sex pheromonal receptors[J].Chinese Journal of Zoology,2013,48(2):298-305.
[6] COLE T B,STACEY N E.Olfactory responses to steroids in an African mouth-brooding cichlid,Haplochromis burtoni (Günther)[J].Journal of Fish Biology,2006,68(3):661-680.
[7] 危起伟,李罗新,杜浩,等.中华鲟全人工繁殖技术研究[J].中国水产科学,2013,20(1):1-11.WEI Q W,LI L X,DU H,et al.Research on technology for controlled propagation of cultured Chinese sturgeon(Acipenser sinensis)[J].Journal of Fishery Sciences of China,2013,20(1):1-11.
[8] 龚全,刘亚,杜军,等.达氏鲟全人工繁殖技术研究[J].西南农业学报,2013,26(4):1710-1714.GONG Q,LIU Y,DU J,et al.Study on artificial reproduction of cultured Acipenser dabryanus Dumeril[J].Southwest China Journal of Agricultural Sciences,2013,26(4):1710-1714.
[9] BRUCH R C,RULLI R D.Ligand binding specificity of a neutral L-amino acid olfactory receptor[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1988,91(3):535-540.
[10] 郭岚,潘萍萍,谭婷,等.超高效液相色谱-荧光法测定大鼠尿液中的黄蝶呤、异黄蝶呤、酪氨酸和色氨酸[J].南昌大学学报(理科版),2014,38(3):218-221.GUO L,PAN P P,TAN T,et al.Determination of xanthopterin,isoxanthopterin,tyrosine and tryptophan in mouse urine by UPLC-LD[J].Journal of Nanchang University (Natural Science),2014,38(3):218-221.
[11] 徐开成,于瑞莲,杨云菲,等.LC-MS/MS法同时测定大鼠血浆中L-色氨酸和L-犬尿氨酸浓度的研究[J].中国药科大学,2016,47(6):714-718.XU K C,YU R L,YANG Y F,et al.Simultaneous determination of L-tryptophan and L-kynurenine in rat plasma by LC-MS/MS[J].Journal of China Pharmaceutical University,2016,47(6):714-718.
[12] 郭岚,孙玄,鄢爱平,等.高效液相色谱-串联质谱法同时测定大鼠尿液中五种可能的癌症标志物[J].分析科学学报,2015,31(5):611-615.GUO L,SUN X,YAN A P,et al.Simultaneous determination of five potential cancer biomarkers in mouse urine by high performance liquid chromatography-tandem mass spectrometry[J].Journal of Analytical Science,2015,31(5):611-615.
[13] 支小平,崔盼盼,柳英霞.高效液相色谱-串联质谱法同时测定人体尿液中的酪氨酸和色氨酸[J].南昌大学学报(理科版),2016,40(4):341-345.ZHI X P,CUI P P,LIU Y X.Simultaneous determination of tyrosine and tryptophan in human urine by HPLC-MS/MS[J].Journal of Nanchang University (Natural Science),2016,40(4):341-345.
[14] 吴智明,张林,李楠.液相色谱-电喷雾串联质谱法分析大肠癌患者尿液色氨酸及其代谢产物[J].重庆医学,2014,43(25):3369-3371.WU Z M,ZHANG L,LI N.Simultaneous determination of tryptophan and its metabolites in urine from colorectal cancer patients by liquid chromatography-electrospray tandem mass spectrometry[J].Chongqing Medicine,2014,43(25):3369-3371.
[15] 郭岚,潘萍萍,鄢爱平,等.UPLC-DAD测定大鼠尿液中的黄蝶呤、异黄蝶呤、酪氨酸和色氨酸[J].分析实验室,2013,32(7):6-9.GUO L,PAN P P,YAN A P,et al.Determination of xanthopterin,isoxanthopterin,tyrosine and tryptophan in mouse urine by UPLC-DAD[J].Chinese Journal of Analysis Laboratory,2013,32(7):6-9.
[16] 李婷婷,刘若男,顾月清.基于比色法快速测定尿液中色氨酸的含量[J].生命科学仪器,2018,16(1):43-47.LI T T,LIU R N,GU Y Q.Rapid determination of tryptophan in urine based on colorimetric method[J].Life Science Instruments,2018,16(1):43-47.
[17] 张带荣.高效液相色谱荧光法测定紫苏子中色氨酸[J].中国医院药学杂志,2008,28(15):1277-1279.ZHANG D R.Quantitative determination of tryptophan in perilla frutescens seeds by HPLC with fluorescence detection[J].Chinese Journal of Hospital Pharmacy,2008,28(15):1277-1279.
[18] 陈军,徐礼生,张兴桃,等.高效液相色谱法测定酶法制备L-色氨酸[J].食品与生物技术学报,2017,36(3):327-330.CHEN J,XU L S,ZHANG X T,et al.Study on high performance liquid chromatography method for determination of enzymatic preparation of L-tryptophan[J].Journal of Food Science and Biotechnology,2017,36(3):327-330.
[19] HUNN J B.Chemical composition of rainbow trout urine following acute hypoxic stress[J].Transactions of the American fisheries society,1969,98(1):20-22.
[20] 邱伟强,刘冰宣,陈舜胜,等.HPLC法快速测水产品中游离芳香族氨基酸含量的研究[J].上海海洋大学学报,2013,22(4):629-633.QIU W Q,LIU B X,CHEN S S,et al.Rapid analysis of free aromatic amino acids in aquatic product by high performance liquid chromatography[J].Journal of Shanghai Ocean University,2013,22(4):629-633.
[21] 任亚萍,唐爱国,周前选,等.高效液相色谱-荧光法同时测定血清中的色氨酸和酪氨酸[J].检验医学,2010,25(10):756-760.REN Y P,TANG A G,ZHOU Q X,et al.High performance liquid chromatography-fluorescence detection for the determination of tryptophan and tyrosine in serum[J].Laboratory Medicine,2010,25(10):756-760.
[22] 穆萨.高效液相色谱-荧光法同时测定重性抑郁障碍患者血清酪氨酸、5-羟色胺和色胺酸[D].长沙:中南大学,2011.MU S.Simultaneous determination of tyrosine,tryptophan and 5-hydroxytryptamine concentrations in serum of major depressive disorder patients by high performance liquid chromatography-fluorescence detection[D].Changsha:Central South University,2011.
[23] 杨永丽,崔盼盼,谭婷,等.高效液相色谱-荧光法同时测定人体尿液中的酪氨酸和色氨酸[J].分析科学学报,2016,32(5):634-638.YANG Y L,CUI P P,TAN T,et al.Simultaneous determination of tyrosine and tryptophan in human urine by HPLC-FLD[J].Journal of Analytical Science,2016,32(5):634-638.
[24] 霍湘,王安利,杨建梅.鱼类对氨基酸的吸收代谢与需求[J].水利渔业,2005,25(3):1-3.HUO X,WANG A L,YANG J M.Absorption,metabolism and demand of amino acids in fish [J].Reservoir Fisheries,2005,25(3):1-3.
[25] FORSTER R P,GOLDSTEIN L.Formation of excretory products[J].Fish Physiology,1969,1:313-350.
[26] SCOTT A P,LILEY N R.Dynamics of excretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate,and of the glucuronides of testosterone and 17β-oestradiol,by urine of reproductively mature male and female rainbow trout(Oncorhynchus myklss)[J].Journal of Fish Biology,1994,44(1):117-129.
[27] YAMBEM H,SHINDO M,YAMAZAKI F.A releaser pheromone that attracts males in the urine of mature female masu salmon[J].Journal of Fish Biology,1999,55(1):158-171.
[2] 甄乾娜.高效液相色谱法分析色氨酸代谢及其在临床诊断中的应用[D].重庆:重庆医科大学,2011.ZHEN Q N.Analysis of application of tryptophan catabolism in clinical diagnosis by HPLC[D].Chongqing:Chongqing University of Medical Sciences,2011.
[3] 赵明江,周小秋.色氨酸对应激诱导鱼类皮质醇分泌的影响[J].水产科学,2007,26(7):420-422.ZHAO M J,ZHOU X Q.The effect of dietary tryptophan on the stress-induced secretion of cortisol[J].Fisheries Science,2007,26(7):420-422.
[4] YAMBE H,KITAMURA S,KAMIO M,et al.L-Kynurenine,an amino acid identified as a sex pheromone in the urine of ovulated female masu salmon[J].Proceedings of the National Academy of Sciences of the United States of America,2006,103(42):15370-15374.
[5] 赖晓健,洪万树,张其永.鱼类嗅觉系统和性信息素受体的研究进展[J].动物学杂志,2103,48(2):298-305.LAI X J,HONG W S,ZHANG Q Y.Research progresses in fish olfactory system and sex pheromonal receptors[J].Chinese Journal of Zoology,2013,48(2):298-305.
[6] COLE T B,STACEY N E.Olfactory responses to steroids in an African mouth-brooding cichlid,Haplochromis burtoni (Günther)[J].Journal of Fish Biology,2006,68(3):661-680.
[7] 危起伟,李罗新,杜浩,等.中华鲟全人工繁殖技术研究[J].中国水产科学,2013,20(1):1-11.WEI Q W,LI L X,DU H,et al.Research on technology for controlled propagation of cultured Chinese sturgeon(Acipenser sinensis)[J].Journal of Fishery Sciences of China,2013,20(1):1-11.
[8] 龚全,刘亚,杜军,等.达氏鲟全人工繁殖技术研究[J].西南农业学报,2013,26(4):1710-1714.GONG Q,LIU Y,DU J,et al.Study on artificial reproduction of cultured Acipenser dabryanus Dumeril[J].Southwest China Journal of Agricultural Sciences,2013,26(4):1710-1714.
[9] BRUCH R C,RULLI R D.Ligand binding specificity of a neutral L-amino acid olfactory receptor[J].Comparative Biochemistry and Physiology Part B:Comparative Biochemistry,1988,91(3):535-540.
[10] 郭岚,潘萍萍,谭婷,等.超高效液相色谱-荧光法测定大鼠尿液中的黄蝶呤、异黄蝶呤、酪氨酸和色氨酸[J].南昌大学学报(理科版),2014,38(3):218-221.GUO L,PAN P P,TAN T,et al.Determination of xanthopterin,isoxanthopterin,tyrosine and tryptophan in mouse urine by UPLC-LD[J].Journal of Nanchang University (Natural Science),2014,38(3):218-221.
[11] 徐开成,于瑞莲,杨云菲,等.LC-MS/MS法同时测定大鼠血浆中L-色氨酸和L-犬尿氨酸浓度的研究[J].中国药科大学,2016,47(6):714-718.XU K C,YU R L,YANG Y F,et al.Simultaneous determination of L-tryptophan and L-kynurenine in rat plasma by LC-MS/MS[J].Journal of China Pharmaceutical University,2016,47(6):714-718.
[12] 郭岚,孙玄,鄢爱平,等.高效液相色谱-串联质谱法同时测定大鼠尿液中五种可能的癌症标志物[J].分析科学学报,2015,31(5):611-615.GUO L,SUN X,YAN A P,et al.Simultaneous determination of five potential cancer biomarkers in mouse urine by high performance liquid chromatography-tandem mass spectrometry[J].Journal of Analytical Science,2015,31(5):611-615.
[13] 支小平,崔盼盼,柳英霞.高效液相色谱-串联质谱法同时测定人体尿液中的酪氨酸和色氨酸[J].南昌大学学报(理科版),2016,40(4):341-345.ZHI X P,CUI P P,LIU Y X.Simultaneous determination of tyrosine and tryptophan in human urine by HPLC-MS/MS[J].Journal of Nanchang University (Natural Science),2016,40(4):341-345.
[14] 吴智明,张林,李楠.液相色谱-电喷雾串联质谱法分析大肠癌患者尿液色氨酸及其代谢产物[J].重庆医学,2014,43(25):3369-3371.WU Z M,ZHANG L,LI N.Simultaneous determination of tryptophan and its metabolites in urine from colorectal cancer patients by liquid chromatography-electrospray tandem mass spectrometry[J].Chongqing Medicine,2014,43(25):3369-3371.
[15] 郭岚,潘萍萍,鄢爱平,等.UPLC-DAD测定大鼠尿液中的黄蝶呤、异黄蝶呤、酪氨酸和色氨酸[J].分析实验室,2013,32(7):6-9.GUO L,PAN P P,YAN A P,et al.Determination of xanthopterin,isoxanthopterin,tyrosine and tryptophan in mouse urine by UPLC-DAD[J].Chinese Journal of Analysis Laboratory,2013,32(7):6-9.
[16] 李婷婷,刘若男,顾月清.基于比色法快速测定尿液中色氨酸的含量[J].生命科学仪器,2018,16(1):43-47.LI T T,LIU R N,GU Y Q.Rapid determination of tryptophan in urine based on colorimetric method[J].Life Science Instruments,2018,16(1):43-47.
[17] 张带荣.高效液相色谱荧光法测定紫苏子中色氨酸[J].中国医院药学杂志,2008,28(15):1277-1279.ZHANG D R.Quantitative determination of tryptophan in perilla frutescens seeds by HPLC with fluorescence detection[J].Chinese Journal of Hospital Pharmacy,2008,28(15):1277-1279.
[18] 陈军,徐礼生,张兴桃,等.高效液相色谱法测定酶法制备L-色氨酸[J].食品与生物技术学报,2017,36(3):327-330.CHEN J,XU L S,ZHANG X T,et al.Study on high performance liquid chromatography method for determination of enzymatic preparation of L-tryptophan[J].Journal of Food Science and Biotechnology,2017,36(3):327-330.
[19] HUNN J B.Chemical composition of rainbow trout urine following acute hypoxic stress[J].Transactions of the American fisheries society,1969,98(1):20-22.
[20] 邱伟强,刘冰宣,陈舜胜,等.HPLC法快速测水产品中游离芳香族氨基酸含量的研究[J].上海海洋大学学报,2013,22(4):629-633.QIU W Q,LIU B X,CHEN S S,et al.Rapid analysis of free aromatic amino acids in aquatic product by high performance liquid chromatography[J].Journal of Shanghai Ocean University,2013,22(4):629-633.
[21] 任亚萍,唐爱国,周前选,等.高效液相色谱-荧光法同时测定血清中的色氨酸和酪氨酸[J].检验医学,2010,25(10):756-760.REN Y P,TANG A G,ZHOU Q X,et al.High performance liquid chromatography-fluorescence detection for the determination of tryptophan and tyrosine in serum[J].Laboratory Medicine,2010,25(10):756-760.
[22] 穆萨.高效液相色谱-荧光法同时测定重性抑郁障碍患者血清酪氨酸、5-羟色胺和色胺酸[D].长沙:中南大学,2011.MU S.Simultaneous determination of tyrosine,tryptophan and 5-hydroxytryptamine concentrations in serum of major depressive disorder patients by high performance liquid chromatography-fluorescence detection[D].Changsha:Central South University,2011.
[23] 杨永丽,崔盼盼,谭婷,等.高效液相色谱-荧光法同时测定人体尿液中的酪氨酸和色氨酸[J].分析科学学报,2016,32(5):634-638.YANG Y L,CUI P P,TAN T,et al.Simultaneous determination of tyrosine and tryptophan in human urine by HPLC-FLD[J].Journal of Analytical Science,2016,32(5):634-638.
[24] 霍湘,王安利,杨建梅.鱼类对氨基酸的吸收代谢与需求[J].水利渔业,2005,25(3):1-3.HUO X,WANG A L,YANG J M.Absorption,metabolism and demand of amino acids in fish [J].Reservoir Fisheries,2005,25(3):1-3.
[25] FORSTER R P,GOLDSTEIN L.Formation of excretory products[J].Fish Physiology,1969,1:313-350.
[26] SCOTT A P,LILEY N R.Dynamics of excretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate,and of the glucuronides of testosterone and 17β-oestradiol,by urine of reproductively mature male and female rainbow trout(Oncorhynchus myklss)[J].Journal of Fish Biology,1994,44(1):117-129.
[27] YAMBEM H,SHINDO M,YAMAZAKI F.A releaser pheromone that attracts males in the urine of mature female masu salmon[J].Journal of Fish Biology,1999,55(1):158-171.
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